Natural killer (NK) cells are a third subtype of lymphocytes besides B and T cells. NK cells provide an important immune function in the defense against viruses and other intracellular pathogens. Unlike B and T cells, NK cells do not exhibit specificity for antigen and they acquired their name because they can kill cells without prior stimulation by antigen. The killing of normal healthy cells is prevented by inhibitory receptors on NK cells that recognize major histocompatibility class I molecules. A large number of receptors that activate the cytotoxic response of NK cells have been described. However, it is not clear if any one of these receptors is sufficient and/or necessary to activate natural cytotoxicity. The major goal of this project is to define the molecular basis of NK cell activation and to characterize the receptors involved. A reconstitution system has been developed to test for the contribution of individual receptors to the activation of NK cell cytotoxicity. Several specific ligands of NK cell receptors were expressed in an insect cell line in order to define the requirements for activation of NK cell cytotoxicity. Surprisingly, expression of ICAM-1 alone was sufficient to induce lysis of the insect cells, suggesting that the integrin LFA-1 is essential not only for adhesion of NK cells to target cells, but also for transmitting activation signals. Expression of ligands for other NK cell activation receptors (such as CD16, CD2, and 2B4) was not sufficient to induce cytotoxicity but co-expression with ICAM-1 did enhance the lysis induced by ICAM-1 alone. The regulation of effector functions in resting NK cells is not well understood. The insect cell system was used to show that engagement of receptors CD2 and 2B4 by their respective ligand on target cells resulted in a strong enhancement of adhesion, even in the absence of cytokines or chemokines. The NK cell receptor KIR2DL4 has the unique property of inducing interferon-gamma production, but not cytotoxicity, by resting NK cells in the absence of cytokines. A DNA microarray study, followed by protein analysis, revealed that KIR2DL4 ligation induces a pro-inflammatory response. Furthermore, KIR2DL4 carries an unusual combination of structural elements that endow it with both activating and inhibitory potential.